Screening of Fungi for the Control of Aspergillus parasiticus

Transcription

1 Screening of Fungi for the Control of Aspergillus parasiticus Dusanee Thanaboripat 1 *, Nannapat Sappakitjanon 1, Luntharima Prommi 1 and Sittichai Chareonsettasilp 2 1 Department of Biology and 2 Department of Statistics, Faculty of Science, King Mongkut s Institute of Technology Ladkrabang, Bangkok 10520, Thailand Abstract Fungal isolates previously selected from soil [KMC 5, SRS 4, I08 (1) and I06 (3)] were screened for their ability to produce antifungal substances against aflatoxin producing Aspergillus parasiticus IMI The production of antifungal substances by these isolates was investigated in yeast extract sucrose broth under static condition. Culture filtrates of these isolates were subsequently tested against A. parasiticus on PDA. The filtrates of all isolates produced varying degrees of inhibition against A. parasiticus with the highest inhibition zone of mm. These isolates were identified as Trichoderma and the other two isolates were Penicillium sp. Keywords: Trichoderma, Aspergillus parasiticus, aflatoxin, biocontrol 1. Introduction Aflatoxins are secondary metabolites produced by certain strains of Aspergillus flavus, A. parasiticus, A. nomius, A. tamarii, A. bombycis and A. pseudotamarii [1-6]. Aflatoxins are one of the most potent toxic substances that occur naturally [7] and their contamination in economic crops is a major problem all over the world [8]. Aflatoxin is a serious human carcinogen and has been identified as a potential biological weapon for food and water contamination [9]. Ingestion of aflatoxins in contaminated food or feed can cause aflatoxicosis. Aflatoxin poisoning has been reported from all parts of world in almost all domestic and non domestic animals like cattle, horses, rabbits, and other non human primates [7]. Diet is the major way through which humans and animals are exposed to aflatoxins. Furthermore, exposure to aflatoxin can be through ingestion of contaminated milk containing aflatoxin M1. Occupational exposures to aflatoxins in agricultural workers, people working in oil mills, and granaries have been reported [10]. Experiments conducted in different areas especially in China and in the African countries have shown high incidence of liver cancer [11,12]. The outbreak of acute aflatoxicosis in Kenya in 2004 was one of the most severe case of human aflatoxin poisoning in history [13] with a case fatality rate of 39%. This epidemic resulted from the ingestion of contaminated maize [14]. Many countries have limitation of aflatoxin residue in agricultural products for safety of consumer. Current legislative level of aflatoxins permissible in Thailand is 20 ng/g or 20 ppb [15] whereas European limit for food is 1-5 µg/kg for aflatoxin B1 and 4-10 µg/kg for total aflatoxins [16]. FAO and WHO have reportd a high incidence of hepatitis B virus infection in areas where dietary exposure to aflatoxins *Corresponding author: Tel: Fax: ktdusane@kmitl.ac.th 95

2 was prevalent. Subsequent research proved that both hepatitis B virus and aflatoxins act synergistically in the imposed regulatory guidelines of 20 ppb of total aflatoxins as the maximum allowable limit in food or feed [17]. In Thailand, the contamination of aflatoxins ranging from µg/kg was found in one hundred and twenty noodle dishes (51%) consumed in 3 areas of Bangkok [18] while 5 herbal medicinal products out of 28 products were also found to be contaminated with detectable amount of aflatoxins ranging from ng/g [15]. Physical, chemical and biological methods have been investigated in order to prevent the growth of aflatoxigenic fungi, to reduce or eliminate the toxin levels, to degrade or detoxify the toxins in foods and feeds [19]. Many microorganisms have mechanisms to compete with others for nutrients and growth development. Fungi have many mechanisms such as growth competition, antibiotic production and endolysis, mycoparasitism and exolysis [20]. Many groups of fungi are well known as biocontrol agents. For example, Trichoderma viride and T. harzianum can produce amylolytic, pectinolytic, proteolytic and cellulolytic enzymes for growth inhibition of A. flavus and Fusarium moniliforme [21]. Corynebacterium rubrum, A. niger, Mucor ambiguus and T. viride can reduce aflatoxin B1 in vitro [22]. Nontoxigenic strains of A. flavus (NRRL 21882) and an orange brown color mutant of A. parasiticus (NRRL 21369), each lacking the ability to produce aflatoxins, had competitive effect and reduced aflatoxin contamination occurred during storage [23]. The extract of P. oxalicum showed fungicidal activity which affected A. candidus, A. versicolor, P. griseofulvum, Curvularia trifolii and Botrytis cinerea [24]. The objective of this study is to screen fungal isolates previously selected from soil [KMC 5, SRS 4, I08 (1) and I06 (3)] with the ability to control Aspergillus parasiticus IMI on PDA. 2. Materials and Methods 2.1 Fungi Aspergillus parasiticus IMI obtained from International Mycological Institute, U.K Fungal isolates KMC 5, SRS 4, I08 (1) and I06 (3) previously isolated from cultivated soil by Wongsariya et al.[25]. 2.2 Cultivation of fungi All fungal isolates were cultured on Potato Dextrose Agar (PDA) for 7 days at room temperature. 2.3 Preparation of spore suspension Spores of fungal isolates were prepared by growing these fungi on PDA for 7 days before harvesting and filtering through sterile cotton wool by sterile distilled water plus 0.02 % tween 80. The numbers of spores were counted by haemacytometer. 2.4 Culture filtrate preparation Yeast extract sucrose (YES) was used for culturing each fungal isolates. The medium was prepared by adding 45 ml broth into 125 ml flask and autoclaved. Spore suspension (10 6 spores/ml) of each isolate was inoculated into each flask and incubated at static condition for 14 days. Culture filtrates of these fungi were then filtered through sterile cotton wool and membrane filter (0.2 µm) under sterile condition to obtain cell-free filtrates [26]. 96

3 2.5 Agar diffusion test Twenty five ml of PDA were poured into a Petri dish (15 cm diameter) and left to set and 20 ml of PDA inoculated with spores of A. parasiticus IMI (10 6 spores/ml) were poured over the surface of PDA agar plate [27]. Sterile cylinder cups (6mm. diameter) containing 300 µl of each culture filtrate were placed onto the same plates. Cylinder cup containing broth without any culture was served as a control. The plates were then incubated for 4 days at room temperature and the inhibition zones were determined on days 2, 3 and 4 using vernier digital caliper. All experiments were repeated at least five times. 2.6 Identification of fungal isolates Fungal isolates were identified by morphological studies using PDA and 2% malt extract agar according to the methods of Rifai [28] and Bissett [29-33]. 3. Results and Discussion The effects of antibiotic substances produced by 4 isolates of fungi, i.e, isolates KMC 5, SRS I08 (1) and I06 (3) on the growth of A. parasiticus IMI on agar plate were given in Table 1 and Figure 1. The results show that the culture filtrate of isolate I06 (3) gave the best inhibitory effect with the inhibition zone of mm followed by the culture filtrates of isolates KMC 5, I08 (1) and SRS 4 with the inhibition zones of , and mm, respectively. The result suggested that these fungal isolates may produce some biological metabolites that affected other fungi. Isolates KMC 5 and SRS 4 were identified as Trichoderma atroviride whereas isolates I08 (1) and I06 (3) were identified as Penicillium sp. (Figures 2 and 3). Our previous investigation showed that Trichoderma isolates TISTR 3167 and C1-1 selected from 16 isolates gave the best inhibitory effect on A. flavus IMI and aflatoxin production [34]. Reddy et al. [35] showed that rice seed treatment with T. virens, Pseudomonas fluorescens, Rhodococcus erythropolis, clove extract or carbendazim prevented or drastically reduced the contamination of Aspergillus and aflatoxin production. Among the biocontrol agents, culture filtrates of Rh. erythropolis completely inhibited the aflatoxin B1 production at 25 ml/kg concentration whereas the other biocontrol agents, Ps. fluorescens, T. virens and Bacillus subtilis showed 93, 80 and 68% reduction of A. flavus growth and 83.7, 72.2 and 58 % reduction of aflatoxin B1 at 200 mg/kg, respectively [36]. The undiluted culture filtrates of T. harzianum and T. pseudo-koningii completely inhibited spore germination of rot pathogens such as Macrophomina phaseolina, Alternaria sp., Fusarium solani and Aspergillus niger whereas 50% dilution gave varying degree of inhibition of spore germination [37]. Possible mechanisms of antagonism by Trichoderma spp. have been suggested to involve the production of antibiotics [38] and/or hydrolytic enzyme [39] as well as parasitism [40]. Production of volatiles and extracellular enzymes rather than mycoparasitism was the likely means by which Trichoderma species suppressed the growth of A. flavus and F. monoliforme [21, 41]. Culture filtrates of Trichoderma viride and T. harzianum were inhibitory of F. moniliforme and, to a lesser extent, A. flavus. The degree of inhibition was dependent on the carbon or nitrogen source incorporated into the medium [28]. Most strains of Trichoderma can produce volatiles and non volatile toxic substances such as harzianic acid, alamethicins and tricholin that hinder growth of other microorganisms [42]. Some aggressive Trichoderma spp. used as biocontrol agents are capable of producing either antibiotics or extracellular enzymes or both [43]. These findings are in agreement with our result. Thus the inhibitory activity of Trichoderma atroviride (isolates KMC 5 and SRS 4) on the growth of A. parasiticus IMI on agar plate might be due to diffusible antibiotic substances or 97

4 extracellular enzymes or volatile compounds produced by Trichoderma isolates. However, their inhibition was less than Penicillium isolate I06 (3). Penicillium spp. can produce secondary metabolites, e.g. Penicillium griseofulvum produces griseofulvin that can inhibit bacterial growth [44] while P. citrinum produces citrinin and Table 1 Effect of culture filtrates of 4 fungal isolates on the growth of A. parasiticus IMI Fungal isolate Diameter of inhibition zone (mm) * I06 (3) KMC I08(1) SRS A. B. Figure 1 Inhibition of A. parasiticus by culture filtrate (antibiotic substance) of Penicilliumisolate I06 (3) in YES (static culture condition). A. 2 day-old culture B. 4 day-old culture A. B. Figure 2 Conidiophores of Penicillium A. Isolate I08 (1) B. Isolate I06 (3) 98

5 A. B. Figure 3 Conidiophores of Trichoderma A. Isolate KMC 5 (1) B. Isolate SRS inhibits the root-rot pathogen, Heterobasidion annosum [45]. Penicillium oxalicum strain PY-1 isolated from soil produced antifungal substances effectively suppressed the mycelia growth of S. sclerotiorum and other tested plant pathogenic fungi[46]. Ma et al. [47] isolated Penicillium striatisporum Pst 10 from the rhizosphere of chilli peppers and tested for antagonistic effects on Phytophthora spp., Cladosporium cucumerium and Sclerotinia sclerotiorum. Pst10 could produce antifungal substances in liquid culture in PDB or solid fermentation on wheat barn. Sterilized liquid culture filtrate (SLCF) of Pst 10 grown in PDB completely inhibited mycelium growth of Ph. capsici. Production of antifungal substances in liquid cultures occurred for approximately 8-10 days when the inhibitory effects on mycelia growth of Ph. capsici reached a maximum. 4. Conclusions This preliminary study showed the biological control mechanism of Penicillium sp. (isolate I06 (3)) and Trichoderma atroviride (isolate KMC 5) against A. parasiticus IMI may be the production of antifungal substances. Although there have been reports on the ability of Trichoderma isolates and their substances to inhibit the growth of Aspergillus sp. but the ability of antimicrobial substances of Penicillium sp. to inhibit the growth of aflatoxin producing fungi have not been reported so far. Thus this may lead to the discovery of new biological control agents for controlling the growth of aflatoxin producing fungi. Further studies have to be conducted in order to identify these antifungal substances. References [1] Goto T., Wicklow, D. T. and Ito, Y Aflatoxin and cyclopiazonic acid production by a sclerotium - producing Aspergillus tamarii strain. Applied and Environmental Microbiology, 62, [2] Peterson, S. W., Ito, Y., Horn, B. W. and Goto, T Aspergillus bombycis, a new aflatoxigenic species and genetic variation in its sibling species, A. nomius. Mycologia, 93,

6 [3] Wilson, D. M. and Payne, G. A Factors affecting Aspergillus flavus group infection and aflatoxin contamination of crops. In: David L. Eaton and John D. Groopman, Ed. The Toxicology of Aflatoxins. Human Health, Veterinary, and Agricultural Significance. San Diego, Academic Press, Inc., pp [4] Ito, Y., Peterson, W., Wicklow, D. T. and Goto, T Aspergillus pseudotamarii, a new aflatoxin producing species in Aspergillus section Flavi. Mycological Research, 105 (2), [5] Carlile, M. J., Watkinson, S. C. and Gooday, G. W The Fungi. 2 nd ed. San Diego, Academic Press. [6] Godet, M. and Manaut, F Molecular strategy for identification in Aspergillus section Flavi. FEMS Microbiological Letter, 304 (2), [7] Bommakanti, A. S. and Waliyar, F Importance of aflatoxins in human and livestock health. [Online]. Available: [8] Smith, J. E. and Moss, M. O Mycotoxins. Formation, Analysis and Significance. Chichester, John Wiley & Sons. [9] Smith, J. E Biotechnology. 4 th ed. Cambridge, Cambridge University Press. [10] Sorenson, W. G, Jones, W., Simpson, J. and Davidson, J. I Aflatoxin in respirable airborne peanut dust. Journal of Toxicology and Environmental Health, 14, [11] Montesano, R., Hainaut, P. and Wild, C. P Hepatocellular carcinoma: From gene to public health. Review Journal of National Cancer Institute, 89, [12] Groopmann, J. D. and Thomas, W. K CRC Critical Reviews in Toxicology, Chapter 19, pp [13] Probst, C., Njapau, H. and Cotty, P. J Outbreak of an acute aflatoxicosis in Kenya in 2004: Identification of the causal agent. Applied Environmental Microbiology, 73 (8), [14] Lewis, L., Onsongo, M., Njapau, H., Rogers, H. S., Luber, G., Kieszak, S., Nyamongo, J., Backer, L., Dahiye, A. M., Misore, A., DeCock, K., Rubin, C. and the Kenya Aflatoxicosis Investigation Group Aflatoxin contamination of commercial maize products during an outbreak of acute aflatoxicosis in Eastern and Central Kenya. Environmental Health Perspective, 113, [15] Tassaneeyakul, W., Razzazi-Fazeli, E., Porasuphatana, S. and Bohm, J Contamination of aflatoxins in herbal medicinal products in Thailand. Mycopathologia, 158, [16] Creppy, E. E Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicology Letters, 127, [17] Krishnamurthy, Y. L. and Shashikala, J Inhibition of aflatoxin B1 production of Aspergillus flavus, isolated from soybean seeds by certain natural plant products. Letters in Applied Microbiology, 43, [18] Promhirangul, P., Srianujata, S., Charoenkiatkul, S. and Kusamran, W Study of aflatoxin content in noodle dishes sold in three areas of Bangkok. M.Sc. Thesis, Graduate School, Mahidol University, Bangkok, Thailand. [19] Thanaboripat, D Importance of aflatoxins. KMITL Science Journal 2 (1), [20] Campbell, R Biological Control of Microbial Plant Pathogens. New York, Cambridge University Press. [21] Calistru, C., Mclean, M. and Berjak, P In vitro studies on the potential for biological control of Aspergillus flavus and Fusarium moniliforme by Trichodema species. I. Macroscopial and microscopical observations of fungal interactions. Mycopathologia, 139,

7 [22] Mann, R. and Rehm, H. J Degradation products from aflatoxin B 1 by Corynebacterium rubrum, Aspergillus niger, Trichoderma viride and Mucor ambiguous. Applied Microbiology and Biotechnology, 2 (4), [23] Dorner, J. W. and Cole, R. J Effect of application of nontoxigenic strains of Aspergillus flavus and A. parasiticus on subsequent aflatoxin contamination of peanuts in storage. Journal of Stored Products Research, 38, [24] Santamarina, M. P., Roselló, J., Llacer, R. and Sanchis, V Antagonistic activity of Penicillium oxalicum Corrie and Thom, Penicillium decumbens Thom and Trichoderma harzianum Rifai isolates against fungi, bacteria and insects in vitro. Review Iberoam Micology, 19, [25] Wongsariya, K., Kuntonwan, S. and Sangvorawanna, S Screening of Trichoderma spp. for inhibition of aflatoxin producing fungi. A Special Project. Faculty of Science, King Mongkut s Institute of Technology Ladkrabang, Bangkok, Thailand. [26] Calistru, C., Mclean, M. and Berjak, P In vitro studies on the potential for biological control of Aspergillus flavus and Fusarium moniliforme by Trichodema species. A study of the production of extracellular metabolites by Trichoderma species. Mycopathologia, 137, [27] Lorian, V Antibiotic in Laboratory Medicine. Philadelphia, Lippincott Williams & Wilins. [28] Rifai, M. A A revision of the genus Trichoderma. Mycol. Paper No 116. Commonwealth Mycological Institute, Kew. [29] Bissett, J A revision of the genus Trichoderma. I. Section Longibarchiatum sect. nov. Canadian Journal of Botany, 62, [30] Bissett, J. 1991a. A revision of the genus Trichoderma. II. Infrageneric classification. Canadian Journal of Botany, 69, [31] Bissett, J. 1991b. A revision of the genus Trichoderma. III. Section Pachybasium. Canadian Journal of Botany, 69, [32] Bissett, J. 1991c. A revision of the genus Trichoderma. IV. Additional notes on section Longibarchiatum. Canadian Journal of Botany, 69, [33] Bissett, J Trichoderma atroviride. Canadian Journal of Botany, 70, [34] Liu, R., Yang, Q., Thanaboripat, D. and Thunsukon, P Screening of Trichoderma species for biological control activity on Aspergillus flavus. KMITL Science Journal, 4 (1), [35] Reddy, C. S., Reddy, K. R. N., Muralidharan, K. and Mangala, U.N Aflatoxigenic Aspergilli and aflatoxin contamination of rice and its management in India. [Online]. Available: posters/ AAA%203rd% 20poster/118.pdf [36] Reddy, K. R. N., Reddy, C. S. and Muralidharan, K Potential of botanicals and biocontrol agents on growth and aflatoxin production by Aspergillus flavus infecting rice grains. Food Control 20, [37] Odebode, A. C Control of postharvest pathogens of fruits by culture filtrate from antagonistic fungi. Journal of Plant Protection Research, 46 (1), 1-5. [38] Ghisalberti, E. I. and Sivasithamparam, K Antifungal antibiotics produced by Trichoderma spp. Soil Biology and Biochemistry, 23, [39] Haran, S., Schickler, A. and Chet, I Differential expression of Trichoderma harzianum chitinases during mycoparasitism. Phytopathology, 86, [40] Benhamou, N. and Chet, I Hyphal interaction between Trichodema harzianum and Rhizoctonia solani: Ultrastructure and gold chemistry of the mycoparasitic process. Phytopathology, 83,

8 [41] Gachomo. E. W. and Kotchoni, S. O The use of Trichoderma harzianum and T. viride as potential biocontrol agents against peanut microflora and their effectiveness in reducing aflatoxin contamination of infected kernels. Biotechnology, 7, 1-9. [42] Vey, A., Hoag, I. R. E. and Butt, T. M Toxic metabolites of fungal biocontrol agents. In: T. M. Butt, C. Jackson and N. Magan, Ed. Fungi as Biocontrol Agents: Progress, Problems and Potential. CAB International Publishing, Bristol, p.311. [43] Benhamou, N. and Cherif, M Cytochemical aspects of chitin breakdown during the parasitic action of Trichodema sp. on Fusarium oxysporum f. sp. radicis-lycopersici. Phytopathology, 71, [44] Bold, H. C., Alexopoulos, C. J. and Delevoryas, T Morphology of Plant and Fungi. 4 th ed. New York. Harper and Row Publisher. [45] Pohanka, A Antifungal Antibiotics from Potential Biocontrol Microorganisms. Doctor s dissertation. Swedish University of Agricultural Science. [46] Yang, L., Xie, J., Jiang, D., Fu, Y., Li, G. and Lin, F Antifungal substances produced by Penicillium oxalicum strain PY-1-potential antibiotics against plant pathogenic fungi. World Journal of Microbiological Biotechnology, 24, [47] Ma, Y., Chang, Zhi-zhou, Zhao, Jiang-tao and Zhou, Ming-guo Antifungal activity of Penicillium stratisporum Pst10 and its biocontrol effect on Phytophthora root rot of chilli pepper. Biological Control, 44 (1),